The functional importance of CD28 in controlling the set point for immune responsiveness has been well established over the past two decades. Initially, the impact of CD28 was attributed to amplification of TCR signaling during activation of nave T cells and generation of effector function. However, CD28 can also enhance Treg development and function, implicating CD28 in the negative regulation of immune responses. We have discovered of a novel mechanism that controls the avidity of CD28 ligand binding. Our results support a model in which inside-out signaling from the TCR induces a rotation of lumenal, ligand binding domains of CD28 around the dimer interface that allows for a switch from monovalent to bivalent binding and a resulting increased avidity for ligand. We have identified a mutation (KK/IP) within the dimer interface that stabilizes CD28 in this high avidity conformation. We have now introduced this mutation into the germline CD28 locus. The overall goal of this proposal is to determine the functional consequences of bypassing the requirement for TCR signaling to express high avidity CD28 ligand binding. We will achieve this goal through two Specific Aims: 1. Determine the functional impact of constitutive high avidity CD28-ligand binding on T cell tolerance and homeostasis. The balance between CD28 and CTLA-4 engagement within both effector T cells and regulatory T cells can control the outcome of the immune response. In this regard, constitutive expression of high avidity CD28 ligand binding may phenocopy loss or diminished CTLA-4 expression that can result in lymphoproliferative disease. In this Aim, we will test this possibility and determine whether the KK/IP mutation enhances homeostatic expansion of effector T cells and/or misregualtion or Tregs. 2. Determine the functional impact of constitutive high avidity CD28-ligand on CD28 costimulation during T cell activation. The most well documented role for CD28 is during costimulation of nave T cells. In addition to initial T cell activation, CD28 costimulation can impact on T cell differentiation into effector T cells, in particular to Tfh cells. In this Aim we will determine the impact of the KK/IP mutation on nave T cell activation and effector cells differentiation using both in vitro and in vivo activation models. 1